Trypanosoma brucei is the causative agent of African human sleeping sickness and the wasting disease Nagana in cattle. The parasite developmentally regulates its mitochondrial function. The regulation of mitochondrial biogenesis is necessary for the parasite's survival and requires the interaction of two genomes (mitochondrial and nuclear) as well as a major RNA modification process in the mitochondrion (RNA editing). While we have some understanding of the regulation of gene expression in the nucleus we know virtually nothing about how gene expression is regulated in the trypanosome mitochondrion. A major hurdle has been the lack of forward and reverse genetics approaches. The overall goal of the research described in this proposal is to develop a transformation system for trypanosome mitochondria using a novel, high throughput method, thereby providing a genetic approach that will allow us to address the regulation of mitochondrial gene expression during the life cycle of the parasite. In order to develop a mitochondrial transformation system we will: (1) determine the requirements for transformation and replication of DNA as well as gene expression in the mitochondrion;(2) develop a positive selection scheme that allows stable transformation of mitochondria;PUBLIC HEALTH RELEVANCE: Trypanosoma brucei is the causative agent of African human sleeping sickness. The development of a mitochondrial transfection system is imperative for the understanding of gene expression in the mitochondrion of this parasite and may lead to the discovery of novel drug targets.